Identificazioni siti di idrolisi di
proteine
Dati:
Conoscenza della sequenza
Amminoacidi identificati sia qualitativamente che quantitativamente
mediante degradazione di Edman su miscele peptidiche della proteina di
interesse
Applicazioni
1.Verifica sperimentale di modelli molecolari
2. Identificazione di agenti idrolitici sconosciuti
Peptide name: proctolin. Sample: 1500 pmoles of the original peptide
Incubation time: 60 minutes at pH 8
1 2 3 4 5
Peptide sequence: R Y L P T
A)
Edman
steps
Residues (pmoles)
D
10
7
5
3
3
1
2
3
4
5
N
5
4
3
2
1
S
11
4
2
1
1
Q
2
1
1
-
T
11
18
103
92
289
G
23
11
7
6
3
E
5
5
3
2
2
A
17
9
5
3
3
H
2
1
-
B)
Residues identified at each step
1
R
Y
L
P
(836)
(293)
(415)
( 32)
2
Y
L
P
T
(873)
(163)
(219)
( 18)
3
L (803)
P (155)
T (103)
4
P (524)
T ( 92)
5
T (289)
C)
Sequences of the fragments present in the mixture
1) R-Y-L-P-T
intact peptide; about 900 pmoles
60%
2) Y-L-P-T
fragment 2-5; about 180 pmoles
12%
3) L-P-T
fragment 3-5; about 250 pmoles
17%
4) P-T
fragment 4-5; about 30 pmoles
2%
Additional free amino acids (first step): R1, Y2, L3
Y
293
873
38
6
3
R
836
58
13
9
9
P
32
219
155
524
98
M
3
1
-
V
9
7
4
2
-
C
7
6
2
-
W
9
7
4
3
2
F
6
2
2
1
1
I
8
7
7
4
3
K
6
2
1
1
1
L
415
163
803
19
6
Peptide name: proctolin.
Sample: 1500 pmoles of the original peptide
Incubation time: 240 minutes at pH 8
1 2 3 4 5
Peptide sequence: R Y L P T
A)
Edman
steps
Residues (pmoles)
D
11
9
5
2
2
1
2
3
4
5
N
7
6
3
3
2
S
17
9
3
1
1
Q
2
2
2
1
-
T
22
36
142
86
79
G
25
14
9
5
3
E
6
4
2
1
2
A
24
14
9
5
3
H
2
1
1
1
1
B)
Residues identified at each step
1
R
Y
L
P
T
(882)
(401)
(632)
( 74)
( 22)
2
Y
L
P
T
(431)
(172)
(334)
( 36)
3
L (275)
P (149)
T (142)
4
P (200)
T ( 86)
5
T (79)
C)
Sequences of the fragments present in the mixture
1) R-Y-L-P-T
intact peptide; about 450 pmoles
30%
2) Y-L-P-T
fragment 2-5; about 190 pmoles
13%
3) L-P-T
fragment 3-5; about 380 pmoles
25%
4) P-T
fragment 4-5; about 50 pmoles
3%
5) T
residue 5; about 30 pmoles
2%
Y
401
431
25
4
2
R
882
64
17
11
8
P
74
334
149
200
65
M
3
1
1
1
-
V
14
9
6
4
2
C
3
1
-
W
5
3
3
3
2
F
8
4
2
2
1
I
9
7
5
3
3
K
8
3
2
2
1
L
632
172
275
9
5
Peptide name: procasomorphin.
Sample: 1500 pmoles of the original peptide
Incubation time: 120 minutes at pH 8
1 2 3 4 5 6 7 8 9 10
Peptide sequence: V Y P F P G P I P K
A)
Edman
steps
Residues (pmoles)
D
8
7
7
5
5
4
1
2
3
4
5
6
N
5
2
2
2
2
1
S
12
4
3
2
2
1
Q
2
2
2
1
1
1
T
7
4
3
3
2
2
G
525
21
11
6
5
4
E
9
4
4
3
3
3
A
16
7
6
4
3
3
H
-
Y
512
31
8
1
1
-
R
7
4
3
2
2
1
P
11
956
45
116
14
12
M
2
1
-
V
896
19
5
3
2
2
C
2
1
-
B)
Residues identified at each step
1
V
Y
F
G
I
K
(896)
(512)
(461)
(525)
( 94)
(124)
2
P (956)
3
I (154)
4
P (116)
5
K ( 56)
C)
Sequences of the fragments present in the mixture
1. Y-P
fragment 2-3; about 500 pmoles
2. F-P
fragment 4-5; about 500 pmoles
3. G-P-I-P-K
fragment 6-10; about 200 pmoles
6. G-P
fragment 6-7; not quantifiable (a value of about 300 pmoles can be deduced)
7. I-P
fragment 8-9; about 100 pmoles
8. K
residue 10; about 150 pmoles
Additional free amino acids (first step): V1 (completely removed)
W
14
13
12
11
10
10
F
461
12
11
6
2
1
I
94
7
154
12
8
4
K
124
13
6
2
56
7
L
9
5
4
4
3
2
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30
Insulin ß-chain sequence: F V N Q H L C* G S H L V E A L Y L V C* G E R G F F Y T P K A
A)
Edman
steps
RESIDUES (pmoles)
D
31
82
41
12
9
9
9
8
8
7
7
6
6
5
5
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
N
88
353
155
18
10
9
7
7
7
8
5
5
6
6
4
S
15
4
47
49
48
32
23
109
45
11
8
8
7
6
6
Q
163
71
278
127
17
9
8
7
5
5
4
4
5
4
4
T
222
324
36
12
8
7
7
6
6
6
6
5
3
3
2
G
14
173
290
753
140
221
687
158
23
11
9
9
8
8
8
E
119
20
58
268
429
41
96
39
23
20
19
27
23
9
6
A
563
200
22
154
213
23
11
58
34
24
17
17
18
18
5
H
58
102
48
128
72
34
20
17
72
24
5
3
3
2
2
Y
1132
453
53
12
9
7
6
5
5
4
3
3
2
2
2
R
10
7
6
5
93
218
29
12
7
6
5
5
2
1
1
P
13
184
386
76
19
14
10
9
8
7
7
8
7
7
7
M
7
5
2
2
1
1
1
-
V
1478
1702
165
24
9
97
52
30
20
25
45
21
7
3
3
C
***
***
***
*
*
***
*
-
W
16
15
16
17
18
15
13
15
14
13
15
16
15
12
13
F
1322
110
17
11
9
7
70
163
17
9
7
6
5
5
4
I
11
9
8
7
6
5
4
5
5
4
5
4
4
4
4
Minimal value useful for the identification at the first step: 20 pmoles
Minimal value useful for the identification at the next steps: 10 pmoles (carryover subtracted)
Minimal value useful for the identification of tryptophan (W): 20 pmoles
Ratio aspartic acid/asparagine: 0,4
Ratio glutamic acid/glutamine : 0,4
B)
Residues identified at each step (carryover subtracted)
1
F
V
N
Q
H
L
C
E
Y
T
A
K
(1322)
(1478)
( 88)
( 163)
( 58)
(2066)
( ***)
( 119)
(1132)
( 222)
( 563)
( 87)
2
V
N
Q
H
L
C
G
A
Y
T
P
(1554)
( 344)
( 55)
( 96)
( 124)
( ***)
( 170)
( 144)
( 340)
( 302)
( 180)
3
N
Q
H
L
C
G
S
P
K
(120)
(271)
( 38)
(105)
(***)
(273)
( 47)
(331)
( 76)
4
Q
H
L
C
G
S
E
K
A
( 99)
(123)
(103)
( *)
(724)
( 44)
(261)
(154)
(152)
5
H
L
C
G
S
E
R
A
( 59)
(495)
( *)
( 65)
( 43)
(403)
( 93)
(198)
6
L
C
G
S
H
R
V
(137)
(***)
(207)
( 27)
( 27)
(209)
( 96)
7
C
G
S
H
L
V
E
F
( *)
(665)
( 20)
( 17)
( 44)
( 42)
( 92)
( 69)
8
G
S
H
L
V
E
A
F
( 89)
(107)
( 15)
( 23)
( 25)
( 29)
( 57)
(156)
9
S
H
L
V
E
A
(34)
(70)
(22)
(17)
(19)
(28)
10
H
L
V
E
A
(17)
(64)
(23)
(18)
(21)
11
L
V
E
A
12
13
14
(26) V (17) E (20) A (16)
(43) E (25) A (16)
(17) A (15)
(15)
K
87
12
77
162
19
9
7
4
2
2
2
2
1
1
1
L
2066
331
138
117
505
187
63
29
25
67
33
7
5
4
4
C)
Sequences of the fragments present in the mixture
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
F-V-N-Q-H-L-C-G-S-H-L-V-E-A
V-N-Q-H-L-C-G-S-H-L-V-E-A
N-Q-H-L-C-G-S-H-L-V-E-A
Q-H-L-C-G-S-H-L-V-E-A
H-L-C-G-S-H-L-V-E-A
L-C-G-S-H-L-V-E-A
C-G-S-H-L-V-E-A
E-A
L-Y
L-V-C-G-E-R-G-F
V-C-G-E-R-G-F
Y-T-P-K-A
T-P-K-A
T-P
K-A
K
A
fragment 1-14; about 150 pmoles
fragment 2-14; about 400 pmoles
fragment 3-14; about 100 pmoles
fragment 4-14; about 150 pmoles
fragment 5-14; about 100 pmoles;
fragment 6-14; about 100 pmoles
fragment 7-14; about 200 pmoles
fragment 13-14; about 150 pmoles
fragment 15-16; about 350 pmoles
fragment 17-24; about 700 pmoles
fragment 18-24; about 300 pmoles
fragment 26-30; about 350 pmoles
fragment 27-30; about 200 pmoles
possible fragment 27-28; not quantifiable
possible fragment 29-30; not quantifiable
possible residue 29; not quantifiable
residue 30; about 600 pmoles
Additional free amino acids (first step): Y26, F25, L17, from F1 to L6
D)
F
1
V
2
N
3
Q
4
H
5
L
6
C
7
G
8
S H L V E A L Y L
9 10 11 12 13 14 15 16 17
V C G E R G F F Y
18 19 20 21 22 23 24 25 26
T P K A
27 28 29 30
Target sequence of the insulin ß-chain: FVNQHLCGSHLVEALYLVCGERGFFYTPKA
Sample : T30 Maximal length of the ambiguous fragments to be not considered = 2
Total fragments : 30
Fragment N. 1 : FVNQHLCGSHLVEA/ Position: 1 - 14 starting from F 1 ending at A 14
Information:
Cut at level of the peptide bond between A 14 and L 15 is confirmed by the presence of a fragment starting from L 15 (N. 18)
N at the step n. 3, Q at the step n. 4, C at the step n. 7, G at the step n. 8, S at the step n. 9, H at the step n. 10, V at the step n. 12, E at the step n. 13, A at the step n.
14 can belong to this fragment only. You can evaluate the amount of the fragment in the mixture by the picomole data of these residues at the corresponding steps
since they are not in common to other fragments.
Details:
Step n. 1 F ( 1322 pmol) is in common to 3 fragments N. 1, N. 2 (F 1-F 1), N. 21 (F 25-F 25)
Step n. 2 V ( 1554 pmol) is in common to 2 fragments N. 1, N. 20 (L17-F24)
Step n. 3 N ( 120 pmol) belongs to this fragment only
Step n. 4 Q ( 99 pmol) belongs to this fragment only
Step n. 5 H ( 59 pmol) is in common to 2 fragments N. 1, N. 12 (L6-L15)
Step n. 6 L ( 136 pmol) is in common to 2 fragments N. 1, N. 12 (L6-L15)
Step n. 7 C (
*
) belongs to this fragment only
Step n. 8 G ( 89 pmol) belongs to this fragment only
Step n. 9 S ( 34 pmol) belongs to this fragment only
Step n. 10 H ( 17 pmol) belongs to this fragment only
Step n. 11 L ( 26 pmol) is in common to 2 fragments N. 1, N. 9 (H5-L15)
Step n. 12 V ( 16 pmol) belongs to this fragment only
Step n. 13 E ( 20 pmol) belongs to this fragment only
Step n. 14 A ( 16 pmol) belongs to this fragment only
Fragment N. 2 : F* Position: 1 - 1 starting from F 1 ending at F 1
Information:
The presence of this fragment has been deduced. Cut at level of the peptide bond between F 1 and V 2 is confirmed by the presence of a fragment starting from V
2 (N. 3)
Details:
Step n. 1 F ( 1322 pmol) is in common to 3 fragments N. 1 (F1-A14), N. 2, N. 21 (F25-F25)
Fragment N. 3 : VNQHLCGSHLVEA/ Position: 2 - 14 starting from V 2 ending at A 14
Information: